The export of mRNA from its site of biogenesis in the nucleus to sites of translation in the cytoplasm is a key step of the gene expression pathway. During export, the mRNP (mRNA plus bound proteins) passes through the central channel of the nuclear pore complex (NPC). S. cerevisiae Dbp5, a DEAD box protein and important conserved mediator of mRNA export, interacts with two proteins located on the cytoplasmic face of the yeast NPC, the cytoplasmic filament nucleoporin, Nup159, and Gle1. Gle1 and inositol hexakisphosphate (IP6), activate the RNA-dependent ATPase of Dbp5. This generates Dbp5-ADP, which can remove bound proteins from mRNP in vitro. Dbp5 is hypothesized to do this in vivo, altering the exported mRNP so that export is irreversible. Understanding Dbp5's interactions with proteins and mRNPs at the NPC and defining its cycle is key to our understanding of gene expression. Three specific aims are proposed for the next project period. (1) Aim 1 is to delineate steps of the Dbp5 cycle. Using a combination of genetic, biochemical and imaging approaches, the interactions of Dbp5 with both Nup159 and Gle1 will be quantitated and the dynamics of these interactions will be measured by using FRAP. The hypothesis that binding to Nup159 is the first step in the Dbp5 cycle and facilitates its interaction with its activator, Gle1, will be tested. These studies will also address whether Dbp5 must cycle through the nucleus to perform its role in mRNA export. Dominant negative (DN) mutants will be characterized and used to determine which functions must be present and absent for a mutant to be DN. Potential new binding partners of Dbp5 will be identified by using mass spectrometry. The functions of the N- and C-terminal regions of Dbp5 will be investigated. (2) Aim 2 is to investigate Gle1's interaction with NPCs. The hypothesis that Gle1 binds to NPCs not only through Nup42 but also through an additional nup will be explored and the predicted binding partner identified. The dynamics of Gle1's interaction with NPCs will be investigated and a set of new ts mutants of Gle1 will be characterized in an attempt to identify a Gle1 that produces a stable protein and whose mutation affects Gle1's function in mRNA export but not protein synthesis. (3) Aim 3 is to define the role of Nup159 in mRNA export. The N-terminus of Nup159 is a seven-bladed betapropeller and Dbp5 binds to blades 1, 6, and 7. The hypothesis that Nup159's N terminus provides a binding site for an additional protein that participates in mRNA export will be investigated using genetics, biochemistry, and mass spectrometry. Nup159 fused to a photoconvertible GFP will be employed to explore the dynamics of Nup159 and cytoplasmic filament association with NPCs.